The spatially and temporally controlled polymerization or actin is required for the motility or cytokinesis of a wide variety of eukaryotic cell types. Arp2/3 complex has been identified as the long-sought nucleator of filaments which provide the free barbed ends required for actin polymerization in vivo. Although several unrelated proteins have been identified which activate Arp2/3 complex nucleation activity, neither the mechanism of this activity nor the manner in which it is controlled physiologically are well understood. Because we have identified multiple phosphorylation states for subunits of Arp2/3 complex, I presently propose to characterize the mechanisms by which phosphorylation of Arp2/3 complex regulate its biochemical interactions and activities. Additionally, I propose to develop a reconstituted minimal actin-based cytoskeleton that is capable of continuously nucleating, assembling, and disassembling crosslinked actin filaments and is subject to direct visualization by light microscopy. Such a system will be an invaluable tool both for testing quantitative models of the interactions of increasingly large numbers of cytoskeletal proteins and for identifying novel factors that play roles in cytoskeletal dynamics.